Endosomes are heterogeneous membrane-bound compartments that segregate endocytosed macromolecules destined to be degraded in the lysosome from molecules that are either recycled back to the cell surface or routed toward other intracellular destinations. The multivesicular body (MVB) is a late endocytic compartment that contains vesicles formed by inward invagination of the endosomal membrane toward the compartmental lumen. Many proteins that control the physiology of eukaryotic organisms are sorted into MVB vesicles, including activated growth factor receptors and stimulatory components of the immune system. The MVB transport pathway is perturbed in a variety of abnormal pathological conditions. Therefore, a mechanistic understanding of MVB biogenesis may ultimately lead to new therapeutic or diagnostic targets for the treatment of human diseases. The long-term objective of this application is to understand the molecular mechanisms of MVB sorting. A genetic strategy has been used to isolate mutations that block the MVB pathway in the budding yeast Saccharomyces cerevisiae. This approach has uncovered the BR01 gene, which encodes a conserved soluble, cytoplasmic protein (Bro1p) that associates with late endosomes. MVB vesicles are absent in yeast mutants in which the BR01 gene has been deleted. The specific aims of this proposal are 1) to define the stage at which Brolp functions in the MVB pathway, 2) to determine the mechanism for association of Brolp with late endosomes, and 3) to identify co-factors that functionally cooperate with Bro1p in the MVB pathway. Mutations will be constructed in the Brolp amino acid sequence, and the consequences of these mutations will be determined using a combination of functional assays, localization studies, and electron microscopy. Biochemical and genetic studies will be used to characterize other factors that participate in the recruitment of Brolp to late endosomes or functionally cooperate with Bro1p in the MVB pathway.